LOCUS       sequence-490863-        7483 bp    DNA     circular SYN 28-OCT-2025
DEFINITION  Express EGFP under CMV promoter. Xho I-CMVp-AgeI-EGFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7483)
  AUTHORS   Kwak CS, Oflaz FE, Qiu J, Wang X
  TITLE     Human stem cell-specific epigenetic signatures control transgene 
            expression.
  JOURNAL   Biochim Biophys Acta Gene Regul Mech. 2024 Dec;1867(4):195063. doi: 
            10.1016/j.bbagrm.2024.195063. Epub 2024 Oct 20.
  PUBMED    39437851
REFERENCE   2  (bases 1 to 7483)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 28, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biochim 
            Biophys Acta Gene Regul Mech."; date: "2024-12"; volume: 
            "1867(4):195063. doi"; pages: " 10.1016/j.bbagrm.2024.195063. Epub 
            2024 Oct 20"
COMMENT     Sequence Label: pRRL_CMVp-EGFP
FEATURES             Location/Qualifiers
     source          1..7483
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        8..234
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             235..415
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    462..587
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1080..1313
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1498..1542
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1809..1926
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     enhancer        1997..2300
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2301..2504
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2455..2475
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    2527..2551
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      2566..2575
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2572..2574,2575..2577,2578..3291)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2572..2574,2575..2577,2578..3288)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2617..2638)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2878..2897)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3225..3246
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    3334..3922
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(3387..3407)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3805..3816)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             4009..4242
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4314..4435
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4351..4370)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4405..4424
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4475..4610
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4630..4649)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4631..4649)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4659..4676)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4659..4675)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4668..4690)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4817..5272
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4904..4923)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5114..5135
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5298..5402
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5403..5471,5472..6263)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(5621..5640)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6434..7022
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6923..6942
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7176..7193
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7310..7331
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(7346..7351,7352..7369,7370..7376)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7384..7400
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     7389..7411
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     7408..7424
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7408..7424
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     7443..7463
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        7445..7463
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
ORIGIN
        1 agcttaatgt agtcttatgc aatactcttg tagtcttgca acatggtaac gatgagttag
       61 caacatgcct tacaaggaga gaaaaagcac cgtgcatgcc gattggtgga agtaaggtgg
      121 tacgatcgtg ccttattagg aaggcaacag acgggtctga catggattgg acgaaccact
      181 gaattgccgc attgcagaga tattgtattt aagtgcctag ctcgatacat aaacgggtct
      241 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      301 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      361 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
      421 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
      481 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
      541 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
      601 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
      661 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
      721 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
      781 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
      841 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
      901 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggagg aggagatatg
      961 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattga accattagga
     1021 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagc agtgggaata
     1081 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgc agcgtcaatg
     1141 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagca gaacaatttg
     1201 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctgggg catcaagcag
     1261 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagct cctggggatt
     1321 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgc tagttggagt
     1381 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtggga cagagaaatt
     1441 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaacca gcaagaaaag
     1501 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattg gtttaacata
     1561 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggctt ggtaggttta
     1621 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggata ttcaccatta
     1681 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaagg aatagaagaa
     1741 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatc tcgacggtat
     1801 cggttaactt ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta
     1861 gacataatag caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa
     1921 aattttatcg atcacgagac tagccatagt aatcaattac ggggtcatta gttcatagcc
     1981 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
     2041 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
     2101 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
     2161 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
     2221 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
     2281 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
     2341 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
     2401 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
     2461 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctctgg ctaactgtcg
     2521 ggatcaacaa gtttgtacaa aaaagcaggc ttcgaaggag atatcgccac catggtgagc
     2581 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     2641 aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg
     2701 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     2761 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     2821 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac
     2881 gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     2941 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag
     3001 tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag
     3061 gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac
     3121 cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc
     3181 acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag
     3241 ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagta aagggcgaat
     3301 tccagcacac tggcggccgt tactagtgtc gacaatcaac ctctggatta caaaatttgt
     3361 gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct
     3421 ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat
     3481 aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg
     3541 gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag
     3601 ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc
     3661 tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg
     3721 tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc
     3781 gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc
     3841 ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc
     3901 tccctttggg ccgcctcccc gcctggaatt cgagctcggt acctttaaga ccaatgactt
     3961 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     4021 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     4081 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     4141 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     4201 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     4261 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     4321 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     4381 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     4441 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4501 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4561 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     4621 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     4681 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4741 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4801 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     4861 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     4921 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     4981 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5041 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5101 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5161 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5221 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     5281 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     5341 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     5401 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     5461 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     5521 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     5581 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     5641 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     5701 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     5761 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     5821 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     5881 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     5941 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     6001 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     6061 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     6121 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     6181 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     6241 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     6301 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     6361 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     6421 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     6481 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     6541 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     6601 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     6661 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     6721 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     6781 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     6841 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     6901 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     6961 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     7021 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     7081 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     7141 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     7201 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     7261 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     7321 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     7381 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     7441 gcgcaattaa ccctcactaa agggaacaaa agctggagct gca
//
